A quantitative shRNA screen identifies ATP1A1 as a gene that regulates cytotoxicity by aurilide B

Abstract

Genome-wide RNA interference (RNAi) with pooled and barcoded short-hairpin RNA (shRNA) libraries provides a powerful tool for identifying cellular components that are relevant to the modes/mechanisms of action (MoA) of bioactive compounds. shRNAs that affect cellular sensitivity to a given compound can be identified by deep sequencing of shRNA-specific barcodes. We used multiplex barcode sequencing technology by adding sample-specific index tags to PCR primers during sequence library preparation, enabling parallel analysis of multiple samples. An shRNA library screen with this system revealed that downregulation of ATP1A1, an α-subunit of Na+/K+ ATPase, conferred significant sensitivity to aurilide B, a natural marine product that induces mitochondria-mediated apoptosis. Combined treatment with ouabain which inhibits Na+/K+ ATPase by targeting α-subunits potentiated sensitivity to aurilide B, suggesting that ATP1A1 regulates mitochondria-mediated apoptosis. Our results indicate that multiplex sequencing facilitates the use of pooled shRNA library screening for the identification of combination drug therapy targets.

Identification of genes involved in the regulation of TERT in hepatocellular carcinoma

“…The pooled shRNA library is a valuable tool for gene screening. It has identified NQQ1 as a potential drug target for host‐directed tuberculosis therapy, ATP1A1 as a gene that regulates aurilide B cytotoxicity, and the deubiquitinating enzyme, USP5, which modulates cell cycle regulators . The shRNA screening system has also identified HIF1α as a TERT regulator using mouse ES cells …”

“…It has identified NQQ1 as a potential drug target for host-directed tuberculosis therapy, ATP1A1 as a gene that regulates aurilide B cytotoxicity, and the deubiquitinating enzyme, USP5, which modulates cell cycle regulators. [34][35][36] The shRNA screening system has also identified HIF1α as a TERT regulator using mouse ES cells. 37 In this study in a HCC cell line, we identified two genes, C15orf55 and C7orf43, as activators of TERT transcription through SP1 and YAP1, respectively.…”

Cellular response to small molecules that selectively stall protein synthesis by the ribosome

“…None of three ribosomal proteins tested (RPL27 or RPS3, RPS19) was found in these immunoprecipitations (Fig 3C and S8 Fig). Interestingly, Liaud et al 13 ASCC2 and ASCC3 can be found to co-fractionate with ribosomes in a sucrose cushion of K562 cellular extracts (Fig 3D). In high-salt sucrose cushions, ASCC2 remains bound to the ribosome, whereas ASCC3 levels are greatly reduced, suggesting that the interaction of ASCC3 with the ribosome is indirect and/or unstable.…”

“…Genomic screens serve as powerful and unbiased tools to identify genetic modifiers of the action of small molecule inhibitors. They can be used to discover proteins involved in an inhibitor's mechanism of action, and to assess possible targets for combination therapy [13,14]. In particular, CRISPR interference screens (CRISPRi) allow robust and highly specific knockdown of gene transcription with minimal off-target effects [15].…”

Systematic Analysis of Drug Vulnerabilities Conferred by Tumor Suppressor Loss

“…Given that there are more than 60 tumor suppressors in the COSMIC Cancer Gene Census (https:// cancer.sanger.ac.uk/census) (Futreal et al, 2004) and more than 20 major types of anticancer mechanisms, it is possible that loss of other tumor suppressors could sensitize cancer cells to certain drugs, thereby providing new angles to specifically attack cancer. Recently, genome-wide RNAi and CRISPR screening have been applied to study resistance mechanisms to several anticancer drugs such as 6-TG, etoposide, and BRAF inhibitor (Koike-Yusa et al, 2014;Shalem et al, 2014;Takase et al, 2017;Wang et al, 2014;Zhu et al, 2019). However, with a few exceptions, the full spectrum of drug vulnerabilities associated with tumor suppressor loss remains largely unknown.…”